In the proposed studies we will utilize a novel, newly-developed, organ culture model to study the process of human squamous epithelial cell invasion under in situ conditions. The organ culture model consists of 2- mm punch biopsy cultures started from healthy neonatal human foreskin (as well as from adult surgical specimen skin). When the tissue pieces are incubated in a serum-free, growth factor-free basal medium, they remain histologically-normal and biochemically active for up to 30 days. However, when the basal medium is supplemented with a combination of exogenous growth factors including epidermal growth factor, insulin and pituitary extract, the basal keratinocytes become pleomorphic and flatten out along the basement membrane. The epithelial cells undergo a hyperproliferative response and strands of epithelial cells migrate down into the space occupied by the mesenchyme. Although initially lined by basement membrane, the basement membrane eventually ruptures and epithelial cells penetrate the mesenchyme. In the proposed studies, this organ culture model will be used to assess the role of serine proteinases and matrix metalloproteinases in the invasion process. In Specific Aim I, studies will focus on serine proteinases. In the first part of the aim, studies will be carried out to assess levels of serine proteinases under conditions in which normal tissue architecture is maintained and under conditions in which invasion occurs. Following this, a number of interventional approaches (broad spectrum serine proteinase inhibitors, plasmin and plasminogen activator inhibitors, monoclonal antibodies, etc.) will be used to block expression of serine proteinases in the organ cultures. The effects of such interventions on invasion will be determined. In Specific Aim II, the focus will be on metalloproteinases. As with serine proteinases, both analytical and interventional studies will be carried out. In this manner, we hope to identify the changes in serine proteinase and metalloproteinase expression that occur in conjunction with induction of invasion. More importantly, by selective proteinase inhibition, we hope to provide definitive evidence for the involvement of specific enzymes or enzyme cascades in the invasion process. By studying the molecular events that accompany and contribute to invasion in this model, we hope to elucidate the process by which human squamous epithelial cell invasion occurs in man under conditions which mimic closely the clinical setting.